Pseudomonas aeruginosa outer membrane protein F produced inEscherichia coli retains vaccine efficacy

Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified fromP. aeruginosa PAO1 reacted with thisE. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive withE. coli strains lacking thePseudomonas protein F gene. The protein F purified from thisE. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized withE. coli K-12 lipopolysaccharide, immunization with theE. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains ofP. aeruginosa. Antisera from mice immunized with theE. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains ofP. aeruginosa and theE. coli strain containing the cloned F gene, but failed to react at these sites in anE. coli strain lacking the F gene. These data demonstrate thatP. aeruginosa protein F produced inE. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of anyP. aeruginosa lipopolysaccharide.     

Protective immunity of <Emphasis Type="Italic">E. coli</Emphasis>-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.     

Lack of development of phagocytic inhibitory activity in serum of cats immunized withEnterobacter cloacae lipopolysaccharide

Sequentially collected sera from cats immunized withEnterobacter cloacae lipopolysaccharide (LPS) were assessed for their effect on phagocytosis by incubating alveolar marcophage monolayers in the presence of³H-labeled bacteria and 5% serum from control or immunized animals. Unlike serum fromPseudomonas aeruginosa LPS-immunized orP. aeruginosa-infected cats in previous studies, which contained phagocytic inhibitory activity specific forP. aeruginosa, serum from 12 of 13 cats immunized withE. cloacae LPS did not contain phagocytic inhibitory activity forE. cloacae orP. aeruginosa. An enzyme-linked immunosorbent assay (elisa) demonstrated a significant rise inE. cloacae LPS specific IgG (peak titer 1:10,240) by week 13 after immunization. This study suggests that long-term immunization withE. cloacae LPS does not result in the induction of macrophage phagocytic inhibitory activity as previously demonstrated withP. aeruginosa LPS immunization.     

Comparison of the protective ability of O and K immunity in mice after immunization withEscherichia coli

The protective abilities ofEscherichia coli O and K immunity against intraperitoneal infection was compared in CBA mice immunized with formalin-killed bacteria. The K13 immunity achieved seemed somewhat more effective than the O6 immunity. This was also reflected in a slightly more rapid clearance of a liveE. coli O6K13 bacteria in K13-immunized animals than in those immunized with O6. However, the protection achieved afterE. coli O6K2 and O2K1 immunization was related more to O immunity than to K immunity.     

Targeting E. coli invasion of the blood–brain barrier for investigating the pathogenesis and therapeutic development of E. coli meningitis

Escherichia coli is the most common Gram‐negative bacillary organism causing neonatal meningitis. Escherichia coli meningitis remains an important cause of mortality and morbidity, but the pathogenesis of E. coli penetration of the blood–brain barrier remains incompletely understood. Escherichia coli entry into the brain occurs in the meningeal and cortex capillaries, not in the choroid plexus, and exploits epidermal growth factor receptor (EGFR) and cysteinyl leukotrienes (CysLTs) for invasion of the blood–brain barrier. The present study examined whether EGFR and CysLTs are inter‐related in their contribution to E. coli invasion of the blood–brain barrier and whether counteracting EGFR and CysLTs is a beneficial adjunct to antibiotic therapy of E. coli meningitis. We showed that (a) meningitis isolates of E. coli exploit EGFR and CysLTs for invasion of the blood–brain barrier, (b) the contribution of EGFR is upstream of that of CysLTs, and (c) counteracting EGFR and CysLTs as an adjunctive therapy improved the outcome (survival, neuronal injury and memory impairment) of animals with E. coli meningitis. These findings suggest that investigation of host factors contributing to E. coli invasion of the blood–brain barrier will help in enhancing the pathogenesis and development of new therapeutic targets for E. coli meningitis in the era of increasing resistance to conventional antibiotics.     

Induction of antimeningitis immunity by the synthetic peptides: I. The immunoactive synthetic fragments of porin A fromNeisseria meningitidis

Fourteen peptides corresponding to sequences of all the exposed and some of the transmembrane protein regions of porin A from the outer membrane ofNeisseria meningitidis strain B:15:P1.7,16 were synthesized. Mice of various lines were immunized with the free peptides not conjugated with any protein carrier. It was shown that the majority of the peptides possess immunogenic properties. Two peptides were identified binding to antibodies present in the serum of mice after meningitis. Protective properties of a number of the synthesized peptides were studied, and three peptide sequences inducing mice protection to an experimental infection withN. meningitidis were identified.     

The influence of artificial colonization withE. coli strain O83 on the intestinal flora in infants

Dominant bacterial strains present in stool (with particular emphasis onE. coli strains) were examined in 4 groups of healthy infants: breast-fed and bottle-fed, colonized withE. coli O83, and control (non-colonized) breast-fed and bottle-fed newborns. The presence of fimbriae was examined by hemagglutination, the P-fimbriae-bearing strains were tested by the PPA latex test. In addition, adherence to cell line HT-29 and serotyping was performed in selected strains. TheE. coli strain O83 was found to possess type 1 fimbriae. Fewer bacterial strains possessing type 1 fimbriae were found inE. coli O83-colonized infants (except the O83 serotype) than in control infants. TheE. coli O83 strain colonized significantly better the breast-fed than the bottle-fed infants; its higher adherence activity was demonstrated even in cell line HT-29. Finally, colonization withE. coli O83 influenced the character of microbial intestinal flora: the frequency of positiveE. coli isolates was significantly higher in colonized (both breast- and bottle-fed) than noncolonized infants.     

Humoral immunity induced in the lower respiratory tract by local immunization with a temperature-sensitive mutant ofPseudomonas aeruginosa

The nature of the humoral immune mechanisms involved in the protection induced after local immunization with a temperature-sensitive (ts) mutant ofPseudomonas aeruginosa was investigated. We had previously shown that intranasal (i.n.) immunization of granulocytopenic mice protected the animals from lethal pulmonary challenge withP. aeruginosa, whereas mice immunized intraperitoneally were unprotected. Intranasal immunization induced high levels of anti-P. aeruginosa IgG and IgA in the lower respiratory tract, whereas only modest levels of IgG (and no IgA) could be detected in lung lavage fluids from mice immunized by the intraperitoneal (i.p.) route with ts mutant E/9/9. Plasma anti-P. aeruginosa IgG levels after i.n. immunization were lower than those observed after i.p. immunization with similar doses of the ts mutant. The main contribution to the protection induced when mice are immunized intranasally appears to be from IgA in the pulmonary secretions, although other immune mechanisms cannot be discounted.     

Expression and Immunogenicity of Enterotoxigenic <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis> Heat-Labile Toxin B Subunit in Transgenic Rice Callus

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G_M1-ganglioside, a receptor for biologically active LTB, was confirmed by G_M1-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G_M1-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.     

Passive immunity to fatal reovirus serotype 3-induced meningoencephalitis mediated by both secretory and transplacental factors in neonatal mice.

The role of passively acquired immunity to reovirus-induced meningoencephalitis in neonatal mice was examined. It was determined that female mice were capable of conferring protection against viral infection and meningoencephalitis in neonates depending on the route by which the dams were immunized and the serotype of the immunizing virus. Female mice immunized with homotypic virus via the oral route developed the most potent response. Infected neonates born and nursed by these females developed no signs of disease, and no virus was recoverable from their small intestines, livers, or brains following infection. Neonates born to females immunized with homotypic virus by the subcutaneous route manifested no evidence of meningoencephalitis or virus dissemination, yet virus was recovered from neonatal intestines. Mice immunized with heterotypic virus by either the subcutaneous or the oral route also conferred protection against disease; however, virus was recovered in small intestines and livers of infected neonates. Based on results from foster-nursing experiments, it appears that factors obtained both during suckling and by transplacental transfer contribute to protection. Passive transfer of reovirus-immune mouse serum also protected neonates from disease. These results demonstrate that passive immune mechanisms can mediate the protection of neonates against reovirus infection and provide further evidence of the importance of the mucosal immune response in protection against pathogens that invade the host via mucosal tissues.     

Incidence of antimicrobial resistance among Enterobacteriaceae isolated in Riyadh

The antimicrobial resistance of 1,018 isolates of Enterobacteriaceae isolated from fecal specimens of the urban population of Riyadh, Saudi Arabia, was studied. Resistance to 1 or more of 10 antimicrobial agents was encountered in 50.2% of the isolates. Of the isolates tested,Escherichia coli (0.8%) andKlebsiella species (1.6%) were found resistant to seven antimicrobial agents simultaneously: ampicillin, chloramphenicol, kanamycin, colistin, streptomycin, tetracycline, and carbenicillin. Resistance to nalidixic acid was encountered in only 0.68% of theE. coli isolates. No isolate was found to be resistant to gentamicin. Eighty-six of the resistant strains were tested for their ability to transfer their resistance. Forty percent were able to do so withE. coli K-12.     

Regulation of Toll‐like receptor 2 interaction with Ecgp96 controls Escherichia coli K1 invasion of brain endothelial cells

The interaction of outer membrane protein A (OmpA) with its receptor, Ecgp96 (a homologue of Hsp90β), is critical for the pathogenesis of Escherichia coli K1 meningitis. Since Hsp90 chaperones Toll‐like receptors (TLRs), we examined the role of TLRs in E. coli K1 infection. Herein, we show that newborn TLR2^?/? mice are resistant to E. coli K1 meningitis, while TLR4^?/? mice succumb to infection sooner. In vitro, OmpA+ E. coli infection selectively upregulates Ecgp96 and TLR2 in human brain microvascular endothelial cells (HBMEC), whereas OmpA? E. coli upregulates TLR4 in these cells. Furthermore, infection with OmpA+ E. coli causes Ecgp96 and TLR2 translocate to the plasma membrane of HBMEC as a complex. Immunoprecipitation studies of the plasma membrane fractions from infected HBMEC reveal that the C termini of Ecgp96 and TLR2 are critical for OmpA+ E. coli invasion. Knockdown of TLR2 using siRNA results in inefficient membrane translocation of Ecgp96 and significantly reduces invasion. In addition, the interaction of Ecgp96 andTLR2 induces a bipartite signal, one from Ecgp96 through PKC‐α while the other from TLR2 through MyD88, ERK1/2 and NF‐κB. This bipartite signal ultimately culminates in the efficient production of NO, which in turn promotes E. coli K1 invasion of HBMEC.     

Cloning of a mercuric ion-resistance operon intoEscherichia coli 5k using the mini-plasmid technique

Summary Mercury resistant transformant clone ofEscherichia coli 5K bearing the recombinant mini-plasmid pHg (13 MDa) was obtained after treatment of the conjugative 60 MDa R plasmid pBH100 (from wild strainE. coli) withPstI followed by fragment ligation (ligase).     

Inhibition of IL-10 production by maternal antibodies against Group B Streptococcus GAPDH confers immunity to offspring by favoring neutrophil recruitment

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')(2) fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10(-/-)) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

Recombinant outer membrane protein F ofPseudomonas aeruginosa elicits antibodies that mediate opsonophagocytic killing,but not complement-mediated bacteriolysis,of various strains ofP. aeruginosa

Recombinant outer membrane protein F ofPseudomonas aeruginosa was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Rats were immunized intramuscularly with 25 g of recombinant protein F adsorbed to aluminum hydroxide adjuvant on days 1, 14, and 28 and then challenged on day 42 via intratracheal inoculation of agar beads containing cells of a clinical isolate ofP. aeruginosa. On day 49 the lungs were examined macroscopically for the presence and severity of lesions and submitted for quantitation of the bacteria present. The recombinant protein F vaccine afforded significant protection against subsequent challenge withP. aeruginosa in the immunized rats, as compared with control rats immunized with bovine serum albumin. Antisera from the recombinant protein F-immunized rats mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells ofP. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant ofP. aeruginosa. The antisera to recombinant protein F did not promote complement-mediated bacteriolysis ofP. aeruginosa. These data demonstrate that recombinantP. aeruginosa protein F has efficacy as a protective vaccine in a rat model of chronic pulmonary infection.     

Antibody prevents the establishment of persistent arenavirus infection in synergy with endogenous T cells.

A cardinal feature of the biology of lymphocytic choriomeningitis virus (LCMV) is its ability to establish persistent infections in mice. Persistence is usually established by infection of the mouse during the in utero or neonatal period. Susceptibility can be extended to the adult by treatment with immunosuppressive agents or by infection with immunosuppressive strains of LCMV. In this study we investigated the capacity of passively acquired anti-LCMV antibodies to prevent the establishment of persistence in both neonatal and adult mice. Suckling BALB/c mouse pups nursed by mothers immunized against LCMV before pregnancy had higher survival rates following infection than controls and withstood challenge doses of up to 400 PFU without becoming persistently infected. To establish that maternal antibody alone and not maternally derived T cells provided this protection, nonimmune mothers were infused with monoclonal anti-LCMV neutralizing antibodies within 24 h after delivering their pups. Pups nursing on these passively immunized mothers were resistant to persistent LCMV infection. The establishment of persistence in adult BALB/c mice by the immunosuppressive, macrophage-tropic LCMV variant, clone 13 was also prevented by prophylactic treatment with anti-LCMV monoclonal antibodies. However, the protection afforded by passively acquired antibody was found to be incomplete if the recipients lacked functional CD8+ T cells. While 65% of neonatal athymic (nu/nu) mice nursed by immune nu/+ dams resisted low-dose viral challenge (25 PFU), the majority of nude pups challenged with high doses of virus (100 PFU) became persistently infected. Also, protection was incomplete in beta2-microglobulin knockout mice, which lack functional CD8+ T cells, suggesting that a cooperative effect was exerted by the combination of neutralizing antibody and endogenous T cells. These results indicate that antibodies provide an effective barrier to the establishment of persistent infections in immunocompetent mice and reaffirm that vaccines which induce strong humoral responses may provide efficient protection against arenavirus infections.     

Establishment and evaluation of detection methods for process-specific residual host cell protein and residual host cell DNA in biological preparation

To establish accurate detection methods of process-specific Escherichia coli residual host cell protein (HCP) and residual host cell DNA (rcDNA) in recombinant biological preparations. Taking the purification process of GLP expressed by E. coli as a specific-process model, the HCP of empty E. coli was intercepted to immunize mice and rabbits. Using IgG from immunized rabbits as the coating antibody and mouse immune serum as the second sandwich antibody, a process-specific enzyme-linked immunosorbent assay (ELISA) for E. coli HCP was established. Targeting the 16S gene of E. coli, ddPCR was used to obtain the absolute copies of rcDNA in samples. Non-process-specific commercial ELISA kit and the process-specific ELISA established in this study were used to detect the HCP in GLP preparation. About 62% of HCPs, which should be process-specific HCPs, could not be detected by the non-process-specific commercial ELISA kit. The sensitivity of established ELISA can reach 338 pg/mL. The rcDNA could be absolutely quantitated by ddPCR, for the copies of rcDNA in three multiple diluted samples showed a reduced gradient. While the copies of rcDNA in three multiple diluted samples could not be distinguished by the qPCR. Process-specific ELISA has high sensitivity in detecting process-specific E. coli HCP. The absolutely quantitative ddPCR has much higher accuracy than the relatively quantitative qPCR, it is a nucleic acid quantitative method that is expected to replace qPCR in the future.     

Production of vanillin from ferulic acid using recombinant strains ofEscherichia coli

Vanillin is one of the world's principal flavoring compounds, and is used extensively in the food industry. The potential vanillin production of the bacteria was compared to select and clone genes which were appropriate for highly productive vanillin production byE. coli. Thefcs (feruloyl-CoA synthetase) andech (enoyl-CoA hydratase/aldolase) genes cloned fromAmycolatopsis sp. strain HR104 andDelftia acidovorans were introduced to pBAD24 vector with P_BAD promoter and were named pDAHEF and pDDAEF, respectively. We observed 160 mg/L vanillin production withE. coli harboring pDAHEF, whereas 10 mg/L of vanillin was observed with pDDAEF. Vanillin production was optimized withE. coli harboring pDAHEF. Induction of thefcs andech genes from pDAHEF was optimized with the addition of 13.3 mM arabinose at 18 h of culture, from which 450 mg/L of vanillin was produced. The feeding time and concentration of ferulic acid were also optimized by the supplementation of 0.2% ferulic acid at 18 h of culture, from which 500 mg/L of vanillin was obtained. Under the above optimized condition of arabinose induction and ferulic acid supplementation, vanillin production was carried out with four different types of media, M9, LB, 2YT, and TB. The highest vanillin production, 580 mg/L, was obtained with LB medium, a 3.6 fold increase in comparison to the 160 mg/L obtained before the optimization of vanillin production.     

Expression fusion immunogen by live attenuated Escherichia coli against enterotoxins infection in mice

Previous epidemiological studies have shown that enterotoxins from enterotoxigenic Escherichia coli (ETEC) appear to be the most important causes of neonatal piglet and porcine post-weaning diarrhoea (PWD). Thus, it is necessary to develop an effective vaccine against ETEC infection. In the present study, the Kil cassette was inserted into the pseudogene yaiT by homologous recombination to create an attenuated E. coli double selection platform O142(yaiT-Kil). After that, PRPL-Kil was replaced with a fusion gene (LTA1-STa₁₃-STb-LTA2-LTB-STa₁₃-STb) to establish oral vaccines O142(yaiT::LTA1-STa₁₃-STb-LTA2-LTB-STa₁₃-STb) (ER-T). Subsequently, BALB/c mice were orally immunized with ER-T. Results showed that serum IgG and faecal sIgA responded against all ETEC enterotoxins and induced F41 antibody in BALB/c mice by orogastrically inoculation with recombinant E. coli ER-T. Moreover, the determination of cellular immune response demonstrated that the stimulation index (SI) was significantly higher in immunized mice than in control mice, and a clear trend in the helper T-cell (Th) response was Th2-cell (IL-4) exceed Th1-cell (IFN-γ).Our results indicated that recombinant E. coli ER-T provides effective protection against ETEC infection.